Fc binding proteins with cysteine in the C-terminal helical region

ABSTRACT

The present invention relates to Fc binding proteins comprising one or more domains with Cysteine in the C-terminal helical region. The invention further relates to affinity matrices comprising the Fc binding proteins of the invention. The invention also relates to a use of these Fc binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc binding proteins of the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national stage filing under 35 U.S. § 371 of Intl.Appln. No. PCT/EP2018/071232, filed Aug. 6, 2018, which claims priorityto European Appln. No. 18163964.2, filed Mar. 26, 2018, European Appln.No. 18154731.6, filed Feb. 1, 2018, and Intl. Appln. No.PCT/EP2017/069976, filed Aug. 7, 2017.

FIELD OF THE INVENTION

The present invention relates to Fc binding proteins comprising one ormore domains having a Cysteine in the C-terminal helical region. Theinvention further relates to affinity matrices comprising the Fc bindingproteins of the invention. The invention also relates to a use of theseFc binding proteins or affinity matrices for affinity purification ofimmunoglobulins and to methods of affinity purification using the Fcbinding proteins of the invention.

BACKGROUND OF THE INVENTION

Many biotechnological and pharmaceutical applications require theremoval of contaminants from a sample containing antibodies. Anestablished procedure for capturing and purifying antibodies is affinitychromatography using the bacterial cell surface Protein A fromStaphylococcus aureus as selective ligand for immunoglobulins (see, forexample, review by Huse et al., J. Biochem. Biophys. Methods 51, 2002:217-231). Wild-type Protein A binds to the Fc region of IgG moleculeswith high affinity and selectivity and is stable at high temperaturesand in a wide range of pH values. Variants of Protein A with improvedproperties such as alkaline stability are available for purifyingantibodies and various chromatographic matrices comprising Protein Aligands are commercially available. However, in particular wild-typeProtein A based chromatography matrices show a loss of binding capacityfor immunoglobulins following exposure to alkaline conditions.

Technical Problems Underlying the Invention

Most large scale production processes for antibodies or Fc-containingfusion proteins use Protein A for affinity purification. However, due tolimitations of Protein A applications in affinity chromatography thereis a need in the art to provide novel Fc binding proteins with improvedproperties that specifically bind to immunoglobulins in order tofacilitate affinity purification of immunoglobulins. To maximallyexploit the value of the chromatographic matrices comprising Fc bindingproteins it is desirable to use the affinity ligand matrices multipletimes. Between chromatography cycles, a thorough cleaning procedure isrequired for sanitization and removal of residual contaminants on thematrix. In this procedure, it is general practice to apply alkalinesolutions with high concentrations of NaOH to the affinity ligandmatrices. Wild-type Protein A domains cannot withstand such harshalkaline conditions for an extended time and quickly lose bindingcapacity for immunoglobulin. Accordingly, there is an ongoing need inthis field to obtain novel Fc binding proteins capable of withstandinglong-term treatment under alkaline conditions. The present inventionprovides Fc binding proteins that are particularly well-suited foraffinity purification of immunoglobulins but overcome the disadvantagesof the prior art. In particular, a significant advantage of the Fcbinding proteins of the invention is their improved stability at high pHfor a prolonged time period without reducing the Fc binding capacitiesin combination with high dynamic binding capacities. Further, the novelFc binding proteins allow an elution of more than 95% at pH of 3.5 orhigher of the bound Fc protein from the Fc binding protein immobilizedto a matrix.

The above overview does not necessarily describe all problems solved bythe present invention.

SUMMARY OF THE INVENTION

A first aspect of the present invention is to provide an Fc bindingprotein suitable for affinity purification. This is achieved with an Fcbinding protein comprising one or more Fc binding domains, wherein atleast one amino acid in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or54 corresponding to SEQ ID NO: 2 is Cysteine. It is preferred that oneor two amino acids in helix 3 are Cysteine. In some embodiments, the Fcbinding protein is comprising one or more Fc binding domains, wherein atleast one domain comprises or essentially consists of or consists of anamino acid sequence of SEQ ID NO: 2 or an amino acid sequence with atleast 89.5% identity thereto wherein at least one amino acid in position40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 2is Cysteine. In some embodiments, at least one domain comprises oressentially consists of or consists of an amino acid sequence of SEQ IDNOs: 3-16, or an amino acid sequence with at least 89.5% identitythereto, respectively, wherein at least one amino acid in position 40,42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NOs: 3-16is Cysteine. In some embodiments, at least one domain comprises oressentially consists of or consists of an amino acid sequence of SEQ IDNOs: 7-8, or an amino acid sequence with at least 89.5% identitythereto, respectively, wherein at least one amino acid in position 40,42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NOs: 7-8is Cysteine. In some embodiments, one or two amino acids in position 43,46, or 47 corresponding to SEQ ID NO: 2 are Cysteine, preferably oneamino acid in position 43, 46, or 47 or two amino acids in position 43and 46 or 43 and 47. In some embodiments, at least one domain comprisesor essentially consists of or consists of an amino acid sequenceselected from the group of SEQ ID NOs: 17-77, 90-99 or an amino acidsequence with at least 89.5% identity thereto. In a second aspect thepresent invention relates to an affinity separation matrix comprisingthe Fc binding protein of the first aspect.

In a third aspect the present invention relates to a use of the Fcbinding protein of the first aspect or of the affinity separation matrixof the second aspect for affinity purification of immunoglobulins orproteins comprising an Fc part of immunoglobulins.

In a fourth aspect the present invention relates to a method of affinitypurification of immunoglobulins or proteins comprising an Fc part ofimmunoglobulins comprising the steps of (a) providing a liquidcontaining an immunoglobulin; (b) providing an affinity separationmatrix comprising an immobilized Fc binding protein of the first aspectcoupled to said affinity separation matrix; (c) contacting said liquidand said affinity separation matrix, wherein said immunoglobulin bindsto said immobilized Fc binding protein; and (d) eluting saidimmunoglobulin from said matrix, thereby obtaining an eluate containingsaid immunoglobulin. This summary of the invention does not necessarilydescribe all features of the present invention. Other embodiments willbecome apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Amino acid sequences of Fc binding domains with Cysteine in thec-terminal helical region (helix 3). The numbers in the top row refer tothe corresponding amino acid position in the Fc binding domain. FIG. 1A.Fc binding domains of SEQ ID NO: 2. FIG. 1B. Examples for Fc bindingdomains with Cys in helix 3.

FIG. 2 . Immobilization of Fc binding domains with Cysteine in helix 3.FIG. 2A. Coupling efficiency of 1614 proteins with Cysteine in positions43, 46, 47, 50, or 58 on Expoxymatrix. Y-axis: coupled amount of proteinin nmol/ml. FIG. 2B. Coupling of cs14 43C and cs14 46C toEpoxy-activated Sepharose 6B matrix. Y-axis: n(protein per ml resin) innmol

FIG. 3 . Caustic stability of Fc binding proteins with Cysteine in helix33. FIG. 3A. Analysis of the alkaline stability of IB14 with Cysteine inpositions 43, 46, 47, 50, or 58. Y-axis: remaining IgG binding activityof the Fc binding protein IB14 and Cys Variants in %. Grey column: IgGbinding activity after 6 h of continuous 0.5 M NaOH treatment. Blackcolumn: IgG binding activity without NaOH. FIG. 3B. Caustic stabilityand DBC 10% of cs14 with Cysteine in positions 43 or 46. Y-axis: DBC 10%(mg/ml). Black: DBC 10% (mg/ml), 0 h NaOH, light grey: DBC 10% (mg/ml),18 h NaOH, dark grey: DBC 10% (mg/ml), 72 h NaOH, The Cys variants werecompared to cs14 with C-terminal Cysteine (after position 58) and toProtein A.

FIG. 4 . The remaining binding capacity (in %) after 6, 24, and 36 hoursof continuous 0.5 M NaOH treatment is shown for cs26 46C, compared tocs26 without Cys in position 46 and compared to wildtype Protein Adomain C. The x-axis shows the time of 0.5 M NaOH incubation in hours.

FIG. 5 . The dynamic binding capacity (DBC; mg/ml) is shown for cs26 46C(monomer and dimer), compared to cs26 without Cys in position 46, andcompared to recombinant wildtype Protein A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Preferably, the terms used herein are consistent with the definitionsprovided in “A multilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughoutthis specification and the claims which follow, unless the contextrequires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps.

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are used interchangeably and intendedto include the plural forms as well and fall within each meaning, unlessthe context clearly indicates otherwise. Also, as used herein, “and/or”refers to and encompasses any and all possible combinations of one ormore of the listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”). The term “about”, as used herein,encompasses the explicitly recited amounts as well as deviationstherefrom of ±10%. More preferably, a deviation 5% is encompassed by theterm “about”. Several documents (for example: patents, patentapplications, scientific publications, manufacturer's specificationsetc.) are cited throughout the text of this specification. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention. Someof the documents cited herein are characterized as being “incorporatedby reference”. In the event of a conflict between the definitions orteachings of such incorporated references and definitions or teachingsrecited in the present specification, the text of the presentspecification takes precedence.

All sequences referred to herein are disclosed in the attached sequencelisting that, with its whole content and disclosure, is a part of thisspecification.

In the context of the present invention, the term “Fc binding protein”or “immunoglobulin-binding protein” or “Ig binding protein” is used todescribe proteins that are capable to specifically bind to the Fc regionof an immunoglobulin. An Fc binding protein comprises at least one “Fcbinding domain” or “immunoglobulin-binding domain” or “Ig bindingdomain” characterized by three-helix bundles of 58 amino acids withhelix 1 from amino acid residues 7-19, helix 2 from amino acid residues23-37, and helix 3 from amino acid residues 40-55. The Fc bindingdomains may comprise deletions, for example up to 6 amino acids at theN-terminus or up to 4 amino acids at the C-terminus, without losing thethree-helix bundle structure.

An “immunoglobulin” as understood herein can include, but is notnecessarily limited to, mammalian IgG, such as for example human IgG₁,human IgG₂, human IgG₄, mouse IgG, rat IgG, goat IgG, bovine IgG, guineapig IgG, rabbit IgG; human IgM, human IgA; and immunoglobulin fragmentscomprising a Fc region. Due to a specific binding to the Fc region, the“Fc binding proteins” or “Ig binding proteins” of the invention arecapable of binding to entire immunoglobulins, and to immunoglobulinfragments comprising a Fc region, fusion proteins comprising an Fcregion of an immunoglobulin, and conjugates comprising an Fc region ofan immunoglobulin. While the Fc binding proteins of the invention hereinexhibit specific binding to the Fc region of an immunoglobulin, it isnot excluded that Fc binding proteins can additionally bind with reducedaffinity to other regions, such as Fab regions of immunoglobulins.

The term “binding” according to the invention preferably relates to aspecific binding. “Specific binding” means that an Fc binding protein oran Fc binding domain binds stronger to an immunoglobulin for which it isspecific compared to the binding to another non-immunoglobulin target.

The term “binding activity” refers to the ability of an Fc bindingprotein of the invention to bind to immunoglobulin. For example, thebinding activity can be determined before and/or after alkalinetreatment. The binding activity can be determined for an Fc bindingprotein or for an Fc binding protein coupled to a matrix, i.e. for animmobilized binding protein. The term “artificial” refers to an objectthat is not naturally occurring, i.e. the term refers to an object thathas been produced or modified by man. For example, a polypeptide orpolynucleotide sequence that has been generated by man (e.g. for examplein a laboratory by genetic engineering, by shuffling methods, or bychemical reactions, etc.) or intentionally modified is artificial.

The term “dissociation constant” or “K_(D)” defines the specific bindingaffinity. As used herein, the term “K_(D)” (usually measured in “mol/L”,sometimes abbreviated as “M”) is intended to refer to the dissociationequilibrium constant of the particular interaction between a firstprotein and a second protein. In the context of the present invention,the term K_(D) is particularly used to describe the binding affinitybetween an Fc binding protein and an immunoglobulin. An Fc bindingprotein of the invention is considered to bind to an immunoglobulin, ifit has a dissociation constant K_(D) to immunoglobulin of at least 1 μMor less, or preferably 100 nM or less, more preferably 50 nM or less,even more preferably 10 nM or less.

The terms “protein” and “polypeptide” refer to any linear molecularchain of two or more amino acids linked by peptide bonds and does notrefer to a specific length of the product. Thus, “peptides”, “protein”,“amino acid chain,” or any other term used to refer to a chain of two ormore amino acids, are included within the definition of “polypeptide,”and the term “polypeptide” may be used instead of, or interchangeablywith any of these terms. The term “polypeptide” is also intended torefer to the products of post-translational modifications of thepolypeptide, including without limitation glycosylation, acetylation,phosphorylation, amidation, proteolytic cleavage, modification bynon-naturally occurring amino acids and similar modifications which arewell-known in the art. Thus, Fc binding proteins comprising two or moreprotein domains also fall under the definition of the term “protein” or“polypeptides”.

The terms “alkaline stable” or “alkaline stability” or “caustic stable”or “caustic stability” (also abbreviated as “cs” herein) refer to theability of the Fc binding protein of the invention to withstand alkalineconditions without significantly losing the ability to bind toimmunoglobulins. The skilled person in this field can easily testalkaline stability by incubating an Fc binding protein with sodiumhydroxide solutions, e.g., as described in the Examples, and subsequenttesting of the binding activity to immunoglobulin by routine experimentsknown to someone skilled in the art, for example, by chromatographicapproaches.

Fc binding proteins of the invention as well as matrices comprising Fcbinding proteins of the invention exhibit an “increased” or “improved”alkaline stability, meaning that the molecules and matricesincorporating said Fc binding proteins are stable under alkalineconditions for an extended period of time relative to a referenceprotein.

The term “parental” in the term “parental protein” or “parental domain”as used herein refers to an Fc binding protein that is subsequentlymodified to generate a variant of said parental protein or domain. Saidparental protein or domain may be an artificial domain (for example, butnot limited to, SEQ ID NO: 78-83), a naturally occurring Staphylococcusaureus Protein A domain (for example, SEQ ID NO: 84 for domain C, SEQ IDNO: 85 for domain B), or a variant or engineered version of a naturallyoccurring Staphylococcus aureus Protein A domain (for example, SEQ IDNO: 86 for domain Z).

The term “variant” as used herein includes an amino acid sequence of anFc binding protein or domain that differs from another amino acidsequence by at least one amino acid substitution, deletion or insertion.These modifications may be generated by genetic engineering or bychemical synthesis or chemical reactions carried out by man. Forexample, SEQ ID NO: 27 (cs26 A460) is a variant of SEQ ID NO: 7 (cs26).

The term “conjugate” as used herein relates to a molecule comprising oressentially consisting of at least a first protein attached chemicallyto other substances such as to a second protein or a non-proteinaceousmoiety.

The term “modification” or “amino acid modification” refers to anexchange, a deletion, or an insertion of an amino acid at a particularposition in a parent polypeptide sequence by another amino acid. Giventhe known genetic code, and recombinant and synthetic DNA techniques,the skilled scientist can readily construct DNAs encoding the amino acidvariants.

The term “substitution” or “amino acid substitution” refers to anexchange of an amino acid at a particular position in a parentpolypeptide sequence by another amino acid. For example, thesubstitution G46C refers to a Fc binding protein, in which the glycineat position 46 is replaced by a cysteine. For the preceding example, 46Crefers to a cysteine at position 46. For the purposes herein, multiplesubstitutions are typically separated by a slash. For example,A1I/S11A/K35R/A46C refers to a variant comprising the combination ofsubstitutions A1I, S11A, K35R, and A46C.

The term “deletion” or “amino acid deletion” refers to the removal of anamino acid at a particular position in a parent polypeptide sequence.

The term “insertions” or “amino acid insertion” refers to the additionof amino acids to the parent polypeptide sequence.

Throughout this description, the amino acid residue position numberingconvention of FIG. 1 is used, and the position numbers are designated ascorresponding to those for example in SEQ ID NOs: 1-16.

The term “amino acid sequence identity” refers to a quantitativecomparison of the identity (or differences) of the amino acid sequencesof two or more proteins. “Percent (%) amino acid sequence identity” or“percent identical” or “percent identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a sequence that are identical with the amino acid residues in thereference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity.

To determine the sequence identity, the sequence of a query protein isaligned to the sequence of a reference protein. Methods for alignmentare well-known in the art. For example, for determining the extent of anamino acid sequence identity of an arbitrary polypeptide relative to areference amino acid sequence, the SIM Local similarity program ispreferably employed (Xiaoquin Huang and Webb Miller (1991), Advances inApplied Mathematics, vol. 12: 337-357), that is freely available (seealso: http://www.expasy.org/tools/sim-prot.html). For multiple alignmentanalysis ClustalW is preferably used (Thompson et al. (1994) NucleicAcids Res., 22(22): 4673-4680). Preferably, the default parameters ofthe SIM Local similarity program or of ClustalW are used, whencalculating sequence identity percentages.

In the context of the present invention, the extent of sequence identityis generally calculated with respect to the total length of theunmodified sequence, if not explicitly stated otherwise. Each amino acidof the query sequence that differs from the reference amino acidsequence at a given position is counted as one difference. The sum ofdifferences is then related to the length of the reference sequence toyield a percentage of non-identity. The quantitative percentage ofidentity is calculated as 100 minus the percentage of non-identity.

As used herein, the phrases “percent identical” or “percent (%) aminoacid sequence identity” or “percent identity”, in the context of twopolypeptide sequences, refer to two or more sequences or subsequencesthat have in some embodiments at least about 80%, in some embodiments atleast 82%, in some embodiments at least 84%, in some embodiments atleast 86%, in some embodiments at least 87%, in some embodiments atleast 89.5%, in some embodiments at least 91%, in some embodiments atleast 93%, in some embodiments at least 94%, in some embodiments atleast 96%, in some embodiments at least 98%, and in some embodiments100% amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. For clarity reasons, forexample a sequence with at least 89.5% identity includes all sequenceswith identities higher than 89.5% identity, e.g. embodiments with atleast 91%, at least 93%, at least 94%, at least 96%, at least 98%, 100%amino acid identity.

The percent identity exists in some embodiments over a region of atleast 50 residue, at least 52 residues, in some embodiments over aregion of at least 53 residues, in some embodiments over a region of atleast 54 residues, in some embodiments over a region of at least 55residues, in some embodiments over a region of at least 56 residues, insome embodiments over a region of at least 57 residues, and in someembodiments over a region of at least 58 residues.

The term “fused” means that the components are linked by peptide bonds,either directly or via peptide linkers.

The term “fusion protein” relates to a protein comprising at least afirst protein joined genetically to at least a second protein. A fusionprotein is created through joining of two or more genes that originallycoded for separate proteins. Thus, a fusion protein may comprise amultimer of identical or different proteins which are expressed as asingle, linear polypeptide As used herein, the term “linker” refers inits broadest meaning to a molecule that covalently joins at least twoother molecules. In typical embodiments of the present invention, a“linker” is to be understood as a moiety that connects an Fc bindingdomain with at least one further Fc binding domain, i.e. a moietylinking two protein domains to each other to generate a multimer. Inpreferred embodiments, the “linker” is a peptide linker, i.e. the moietylinking the two protein domains is one single amino acid or a peptidecomprising two or more amino acids.

The term “chromatography” refers to separation technologies which employa mobile phase and a stationary phase to separate one type of molecules(e.g., immunoglobulins) from other molecules (e.g. contaminants) in thesample. The liquid mobile phase contains a mixture of molecules andtransports these across or through a stationary phase (such as a solidmatrix). Due to the differential interaction of the different moleculesin the mobile phase with the stationary phase, molecules in the mobilephase can be separated.

The term “affinity chromatography” refers to a specific mode ofchromatography in which a ligand coupled to a stationary phase interactswith a molecule (i.e. immunoglobulin) in the mobile phase (the sample)i.e. the ligand has a specific binding affinity for the molecule to bepurified. As understood in the context of the invention, affinitychromatography involves the addition of a sample containing animmunoglobulin to a stationary phase which comprises a chromatographyligand, such as an Fc binding protein of the invention.

The terms “solid support” or “solid matrix” are used interchangeably forthe stationary phase.

The terms “affinity matrix” or “affinity separation matrix” or “affinitychromatography matrix”, as used interchangeably herein, refer to amatrix, e.g. a chromatographic matrix, onto which an affinity ligande.g., an Fc binding protein of the invention is attached. The ligand(e.g., Fc binding protein) is capable of specific binding to a moleculeof interest (e.g., an immunoglobulin as defined above) which is to bepurified or removed from a mixture.

The term “affinity purification” as used herein refers to a method ofpurifying immunoglobulins as defined above from a liquid by binding theimmunoglobulins as defined above to an Fc binding protein that isimmobilized to a matrix. Thereby, all other components of the mixtureexcept immunoglobulins are removed. In a further step, the boundimmunoglobulin is eluted in purified form.

EMBODIMENTS OF THE INVENTION

The present invention will now be further described. In the followingpassages different aspects of the invention are defined in more detail.Each aspect defined below may be combined with any other aspect oraspects unless clearly indicated to the contrary. In particular, anyfeature indicated as being preferred or advantageous may be combinedwith any other feature or features indicated as being preferred oradvantageous.

In a first aspect the present invention is directed to a Fc bindingprotein, wherein an Fc binding protein comprises one or more domains,wherein at least one amino acid in position 40, 42, 43, 46, 47, 49, 50,51, 53, or 54 corresponding to SEQ ID NO: 2 is Cysteine, preferablywherein one amino acids selected from the group of positions 40, 42, 43,46, 47, 49, 50, 51, 53, and 54 corresponding to SEQ ID NO: 2 areCysteine. In one embodiment of the first aspect the amino acid inposition 43, the amino acid in position 46, the amino acid in position47, the amino acid in position 50, the amino acid in position 51, or theamino acid in position 53 corresponding to SEQ ID NO: 2 is Cysteine. Ina first aspect, the Fc protein comprises one or more domains, wherein atleast one domain comprises or essentially consists of or consists of anamino acid sequence of SEQ ID NO: 2 or an amino acid with at least 80%,at least 81%, at least 82%, at least 84%, at least 86%, at least 87%, atleast 89.5%, at least 91%, at least 93%, at least 94%, at least 96%, atleast 98%, or 100% identity wherein at least one amino acid in position40, 42, 43, 46, 47, 50, 51, 53, or 54 corresponding to SEQ ID NO: 2 isCysteine. It is preferred that not more than 2 Cysteine residues are inhelix 3 of a SEQ ID NO: 2-86, 90-99 or an amino acid sequence with atleast 89.5% identity thereto.

Fc binding domains of the invention are understood as three-helixbundles of typically 58 amino acids with helix 1 from amino acidresidues 7-19, helix 2 from amino acid residues 23-37, and helix 3 fromamino acid residues 40-55. Fc binding is mediated by helix 1 and helix2. The surprising advantage of the Fc binding proteins having Cysteinein helix 3 is that they confer an alkali stability for a prolongedperiod of time, without impairing the Fc-binding properties, and withsite directed coupling efficiencies to a matrix providing a high bindingcapacity. The Fc binding proteins of the invention have less than a 20%reduction in binding capacity following an incubation in 0.5 M NaOH forat least 6 hours up to at least 72 hours. This feature is important forchromatography approaches with cleaning procedures using alkalinesolutions with high NaOH concentrations to remove contaminants on thematrix so for example that the matrix can be used several times.Further, in addition to having high caustic stability, Fc bindingproteins having Cys in helix 3 show high coupling efficiencies. Forexample, the dynamic binding capacity of variants with Cys in, forexample, position 46 is superior compared to recombinant Protein A (seeFIG. 5 ).

Preferred Fc Binding Proteins with Cysteine in Helix 3.

Some embodiments relate to sequences with an amino acid selected fromthe group consisting of SEQ ID NOs: 1-86, 90-99. Some embodiments relateto amino acid sequences with at least 80%, at least 81%, at least 82%,at least 84%, at least 86%, at least 87%, at least 89.5%, at least 91%,at least 93%, at least 94.5%, at least 96%, at least 98%, or 100%sequence identity to an amino acid selected from the group consisting ofSEQ ID NOs: 1-86, 90-99 provided that they have at least one Cysteine inposition 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54. Preferred areembodiments with sequences with at least 89.5% identity to an amino acidselected from the group consisting of SEQ ID NOs: 1-86, 90-99 having atleast one Cysteine in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or54, preferably in position 43 or/and 46, more preferably in position 46.Preferred are embodiments of Fc binding proteins with one or twoCysteines in helix 3.

The amino acid sequences of Fc binding proteins of the invention mightcomprise further modifications, such as insertions, deletions, orfurther substitutions. In some embodiments, Fc binding domains have 1,2, 3, 4, 5, or 6 further substitutions. In some embodiments, Fc bindingdomains have a deletion of 1, 2, 3, or 4 amino acids within the first 4amino acids of its N-terminus and/or a deletion of 1 or 2 amino acids atthe C-terminus. In some embodiments, Fc binding domains have deletionsat the N-terminus, for example in positions 1, 2, and 4, or in positions1, 2, and 3. In some embodiments, Fc binding domains have deletions atthe C-terminus, for example in positions 57 and/or 58 (as shown forexample, but not limited to, SEQ ID NOs: 33-34). Some embodiments relateto amino acid sequences with at least 89.5% sequence identity to theamino acid sequence to any of the afore-mentioned SEQ ID NOs (forexample but not limited to SEQ ID NOs: 1-86, 90-99). Examples for Fcbinding domains with at least one Cys, preferably one Cys, in position40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 include for example but arenot limited to the amino acid sequences shown in FIG. 1 .

SEQ ID NO: 7 (cs26) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 7 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:7 include but are not limited to cs24 (SEQ ID NO: 8). Examples forvariants of cs26 having Cys in position 40, 42, 43, 46, 47, 49, 50, 51,53, or 54 include for example but are not limited to SEQ ID NOs: 26-39,90-99.

SEQ ID NO: 8 (cs24) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 8 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:8 include but are not limited to cs26 (SEQ ID NO: 7). Examples forvariants of cs24 having Cys in position 40, 42, 43, 46, 47, 49, 50, 51,53, or 54 include for example but are not limited to SEQ ID NOs: 26-39,90-99.

SEQ ID NO: 3 (cs14) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 3 or of a sequence withat least 89.5% sequence identity thereto suitable for modifications inposition 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:3 include but are not limited to SEQ ID NO: 10 (cs25), SEQ ID NOs: 11(cs47h3), SEQ ID NO: 12 (cs47h4), SEQ ID NO: 13 (cs74h1), and SEQ ID NO:14 (cs74h2). Examples for variants of cs14 having Cys in position 40,42, 43, 46, 47, 49, 50, 51, 53, or 54 include for example but are notlimited to SEQ ID NOs: 17-20, 40-52, 64, 65, 70, 71, 74-76.

SEQ ID NO: 4 (cs27) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 4 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine.Examples for variants of cs27 having Cys in position 40, 42, 43, 46, 47,49, 50, 51, 53, or 54 include for example but are not limited to SEQ IDNOs: 21-25.

SEQ ID NO: 5 (cs20) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 5 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:5 include but are not limited to SEQ ID NO: 9 (cs17). Examples forvariants of cs20 having Cys in position 40, 42, 43, 46, 47, 49, 50, 51,53, or 54 include for example but are not limited to SEQ ID NOs: 53-57.

SEQ ID NO: 6 (cs42) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 6 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:16 include but are not limited to cs28 (different in position 4) or cs41(SEQ ID NO: 15). Examples for variants of cs42 having Cys in position40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 include for example but arenot limited to SEQ ID NOs: 58-63.

SEQ ID NO: 16 (cs43) and Variants.

In some embodiments, the Fc binding protein is comprising one or moredomains of an amino acid sequence of SEQ ID NO: 16 or of an amino acidsequence with at least 89.5% identity thereto suitable for modificationsin position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 with Cysteine. Forexample, amino acid sequences with at least 89.5% identity to SEQ ID NO:16 include but are not limited to cs44 (different in position 44), cs31(different in positions 25 and 54), or cs45 (different in positions 25and 26). Examples for variants of cs43 with Cys in position 40, 42, 43,46, 47, 49, 50, 51, 53, or 54 include for example but are not limited toSEQ ID NOs: 66-69, 72.

Sequences of Fc Binding Proteins; Preferred Amino Acid Positions.

Surprisingly, Cysteine in helix 3 of Fc binding domains increases thealkaline stability of Fc binding domain compared to a domain withoutCysteine in helix 3 as shown in the Figures and in the Examples, andimproves the site-specific coupling of the Fc binding protein to amatrix which improves capacity.

In some embodiments, said Fc binding domain comprises an Isoleucine atposition 1. It is preferred that the amino acid in position 1 of the Fcbinding domain is not Threonine (T). It is preferred that the amino acidin position 1 is Isoleucine (I) or Alanine (A). Alternatively, position1 may be deleted. In some embodiments, said Fc binding domain comprisesan Alanine (A), Glutamic Acid (E), or Isoleucine (I) at position 11. Itis preferred that the amino acid in position 11 is not Asparagine (N) orLysine (K). It is preferred that the amino acid in position 11 isAlanine (A), Isoleucine (I), Glutamic acid (E), Histidine (H), orProline (P), more preferred A, I, or E, most preferred A. Alternatively,the amino acid in position 11 is Serine (5). In some embodiments, saidFc binding domain comprises an Arginine (R) or Isoleucine (I) atposition 35. It is preferred that the amino acid in position 35 is notProline (P), Asparagine (N), Glycine (G), Tryptophan (W), Alanine (A),Glutamine (Q), or Methionine (M). In some embodiments, said Fc bindingdomain comprises a Leucine (L) at position 42. It is preferred that theamino acid in position 42 is not Tyrosine (Y). In some embodiments, inaddition to 40C, 42C, 43C, 46C, 47C, 49C, 50C, 510, 53C, or 54C, the Fcbinding domain comprises 2, 3 or 4 of the amino acid positions selectedfrom the group consisting of 1I, 11A, 115, 35R, and 42L. In someembodiments, at least 90% of positions 1I, 3A, 6D, 9Q, 10Q, 12A, 13F,14Y, 15E, 161, 17L, 18H, 19L, 20P, 21N, 22L, 23T, 24E, 26Q, 27R, 28N,29A, 30F, 31I, 32Q, 33S, 34L, 36D, 37D, 38P, 39S, 41S, 42L, 45L, 48A,52N, 55Q, 56A, 57P are identical in Fc binding domains of the invention.It is preferred that position 2 is A or D, position 4 is K or Q,position 5 is H or F, position 7 is K or E, position 8 is D, A or E,position 11 is A, S, I, or E, position 25 is D or E, position 35 is R orI, position 40 is V, T, Q, or C, position 42 is L or C, position 43 isE, S or C, position 44 is 1, V or L, position 46 is C, A or G, position47 is E or C, position 49 is K, Q, or C, position 50 is K or C, position51 is L or C, position 53 is D or E or C, position 54 is A, S, or C, andposition 58 is P or K.

An Fc binding protein of the invention comprises one or more Fc bindingdomains that comprises or essentially consists or consists of the aminoacid sequence of SEQ ID NO: 2 or at least 89.5% identical amino acidsequences thereto. The amino acid sequence SEQ ID NO: 2 is the followingamino acid sequence:IX₂AX₄X₅DX₇X₈QQX₁₁AFYEILHLPNLTEX₂₅QRNAFIQSLX₃₅DDPSX₄₀SLX₄₃X₄₄LX₄₆X₄₇AX₄₉X₅₀X₅₁NX₅₃X₅₄QAPX₅₈wherein the amino acid at position 1 is selected from I, or is deleted,the amino acid at position 2 (X₂) is selected from A or D, or isdeleted, the amino acid at position 3 is selected from A, or is deleted,the amino acid at position 4 (X₄) is selected from K or Q, or isdeleted, the amino acid at position 5 (X₅) is selected from H or F, theamino acid at position 7 (X₇) is selected from K or E, the amino acid atposition 8 (X₈) is selected from D, A, or E, the amino acid at position11 (X₁₁) is selected from A, S, 1, or E, the amino acid at position 25(X₂₅) is selected from D or E, the amino acid at position 35 (X₃₅) isselected from R or I, the amino acid at position 40 (X₄₀) is selectedfrom Q, T, V, or C, the amino acid at position 42 (X₄₂) is selected fromL or C, the amino acid at position 43 (X₄₃) is selected from E, S, or C,the amino acid at position 44 (X₄₄) is selected from I, L, or V, theamino acid at position 46 (X₄₆) is selected from G, A or C, the aminoacid at position 47 (X₄₇) is selected from E or C, the amino acid atposition 49 (X₄₉) is selected from K or Q or C, the amino acid atposition 50 (X₅₀) is K or C, the amino acid at position 51 (X₅₁) is L orC, the amino acid at position 53 (X₅₃) is selected from D, E or C, theamino acid at position 54 (X₅₄) is selected from A or S or C, the aminoacid at position 57 is selected from P, or deleted, and the amino acidat position 58 (X₅₈) is selected from P or K, or deleted. Fc bindingproteins of the invention have a cysteine in one or two of the followingpositions: 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54. Selected examplesfor Fc binding domains of the invention include for example but are notlimited to SEQ ID NOs: 17-73, 90-99.

High Alkaline Stability as Result of Cysteine in the c-Terminal Regionof Fc Binding Proteins.

In some embodiments, Fc binding proteins with Cysteine in helix 3provide surprisingly particularly good alkaline stability of the Fcbinding protein, as shown in the Examples and in the Figures. It wasmost surprising and unexpected that the Fc binding proteins withCysteine in helix 3 are able to bind to Ig even after alkaline treatmentfor several hours. The alkaline stability of the Fc binding protein isdetermined by comparing the loss in Ig binding activity after at least 6h incubation in 0.5 M NaOH (see FIG. 3 ). For example, the bindingcapacity of cs26 46C remains at least 20% higher than wildtype domain Cafter prolonged (for example, 36 h) incubation with 0.5 M NaOH (see FIG.4 ).

Affinity to Immunoglobulin.

All Fc binding proteins of the invention bind to Immunoglobulin with adissociation constant K_(D) preferably below 500 nM, or below 100 nM,even more preferably 10 nM or less. Methods for determining bindingaffinities of Fc binding proteins or domains, i.e. for determining thedissociation constant K_(D), are known to a person of ordinary skill inthe art and can be selected for instance from the following methodsknown in the art: Surface Plasmon Resonance (SPR) based technology,Bio-layer interferometry (BLI), enzyme-linked immunosorbent assay(ELISA), flow cytometry, isothermal titration calorimetry (ITC),analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) andenhanced chemiluminescence (ECL). Some of the methods are describedfurther in the Examples. Typically, the dissociation constant K_(D) isdetermined at 20° C., 25° C., or 30° C. If not specifically indicatedotherwise, the K_(D) values recited herein are determined at 22° C.+/−3°C. by surface plasmon resonance. In an embodiment of the first aspect,the Fc binding protein has a dissociation constant K_(D) to human IgG₁in the range between 0.1 nM and 100 nM, preferably between 0.1 nM and 50nM.

Multimers.

In one embodiment of the invention, the Fc binding protein comprises 1,2, 3, 4, 5, 6, 7, or 8, preferably 2, 3, 4, 5, or 6, Fc binding domainslinked to each other, i.e. the Fc binding protein can be, for example, amonomer, a dimer, a trimer, a tetramer, a pentamer, or a hexamer. Amultimer may comprise two, three, four, or even more binding domains.Multimers of the invention are fusion proteins generated artificially,generally by recombinant DNA technology well-known to a skilled person.

In some embodiments, the multimer is a homo-multimer, e.g. the aminoacid sequences of all Fc binding domains of the Fc binding protein areidentical.

A multimer may comprise two or more Fc binding domains, wherein said Fcbinding domains preferably comprise or essentially consist of a sequenceas described above, provided that they have at least one Cysteine inhelix 3.

For example, SEQ ID NO: 27 or SEQ ID NO: 22 were used to generate thehomo-multimeric fusion constructs described herein in Example 1, see forexample SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 25.

In some embodiments, the multimer is a hetero-multimer, e.g. at leastone alkaline stable Fc binding domain has a different amino acidsequence than the other Fc binding domains within theimmunoglobulin-binding protein.

Linker.

In some embodiments of the first aspect, the one or more Fc bindingdomains are directly linked to each other. In other embodiments, the oneor more Fc binding domains are linked to each other with one or morelinkers. Preferred in these typical embodiments are peptide linkers.This means that the peptide linker is an amino acid sequence thatconnects a first Fc binding domain with a second Fc binding domain. Thepeptide linker is connected to the first Fc binding domain and to thesecond Fc binding domain by a peptide bond between the C-terminal andN-terminal ends of the domains, thereby generating a single, linearpolypeptide chain. The length and composition of a linker may varybetween at least one and up to about 30 amino acids. More specifically,a peptide linker has a length of between 1 and 30 amino acids; e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids. It is preferred that theamino acid sequence of the peptide linker is stable against causticconditions and proteases. Linkers should not destabilize theconformation of the domains in the Fc binding protein. Well-known arelinkers that comprise or consist of small amino acids such as glycineand serine. The linkers can be glycine-rich (e.g., more than 50% of theresidues in the linker can be glycine residues). Also preferred arelinkers that comprise further amino acids. Other embodiments of theinvention comprise linkers consisting of alanine, proline, and serine.Other linkers for the fusion of proteins are known in the art and can beused. In some embodiments, the multimer of Fc binding proteins comprisesone or more linkers connecting the Fc binding domains wherein the linkerare identical or different.

Conjugation to a Solid Support.

In some embodiments of the invention, the Fc binding protein isconjugated to a solid support. Cysteine in helix 3 of the Fc bindingdomain comprises an attachment site for site-specific covalent couplingof the Fc binding protein to a solid support. At least one Cysteine inposition 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 enables specificchemical reactions with a reactive group of the solid phase or a linkerbetween the solid phase and the protein, for example selected fromN-hydroxysuccinimide, iodacetamide, maleimide, epoxy, or alkene groups.

In some embodiments of the invention, the Fc binding protein may alsocomprise additional amino acid residues at the N- and/or C-terminal end,such as for example an additional sequence with or without a tag at theN- and/or C-terminal end.

Affinity Separation Matrix.

In another aspect the present invention is directed to an affinityseparation matrix, comprising an Fc binding protein of the first aspect.

In preferred embodiments of the second aspect, the affinity separationmatrix is a solid support. The affinity separation matrix comprises atleast one Fc binding protein of the invention.

An affinity matrix is useful for separation of immunoglobulins andshould retain the Ig binding property even after highly alkalineconditions as applied during cleaning processes. Such cleaning ofmatrices is essential for long-term repeated use of matrices.

Solid support matrices for affinity chromatography are known in the artand include for example but are not limited to, agarose and stabilizedderivatives of agarose (e.g. Sepharose 6B, Praesto™Pure; CaptivA®,rPROTEIN A Sepharose Fast Flow, Mabselect®, PrismA®, and other),cellulose or derivatives of cellulose, controlled pore glass (e.g.ProSep® vA resin), monolith (e.g. CIM® monoliths), silica, zirconiumoxide (e.g. CM Zirconia or CPG®), titanium oxide, or synthetic polymers(e.g. polystyrene such as Poros 50A or Poros MabCapture® A resin,polyvinylether, polyvinyl alcohol, polyhydroxyalkyl acrylates,polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamidesetc) and hydrogels of various compositions. In certain embodiments thesupport comprises a polyhydroxy polymer, such as a polysaccharide.Examples of polysaccharides suitable for supports include but are notlimited to agar, agarose, dextran, starch, cellulose, pullulan, etc, andstabilized variants of these.

The formats for solid support matrices can be of any suitable well-knownkind. Such solid support matrix for coupling the Fc binding protein ofthe invention might comprise for example, one of the following: columns,capillaries, particles, membranes, filters, monoliths, fibers, pads,gels, slides, plates, cassettes, or any other format commonly used inchromatography and known to someone skilled in the art.

In one embodiment, the matrix is comprised of substantially sphericalparticles, also known as beads, for example Sepharose or Agarose beads.Suitable particle sizes may be in the diameter range of 5-500 μm, suchas 10-100 μm, such as 20-80 μm, such as 40-70 μm. Matrices in particleform can be used as a packed bed or in a suspended form includingexpanded beds.

In an alternative embodiment, the solid support matrix is a membrane,for example a hydrogel membrane. In some embodiments, the affinitypurification involves a membrane as matrix to which the Fc bindingprotein of the first aspect is covalently bound. The solid support canalso be in the form of a membrane in a cartridge.

In some embodiments, the affinity purification involves a chromatographycolumn containing a solid support matrix to which the Fc binding proteinof the first aspect is covalently bound. The Fc binding protein of theinvention may be attached to a suitable solid support matrix viaconventional coupling techniques. Methods for immobilization of proteinligands to solid supports are well-known in this field and easilyperformed by the skilled person in this field using standard techniquesand equipment.

Use of the Fc Binding Protein.

In a third aspect the present invention is directed to the use of the Fcbinding protein of the first aspect or an affinity matrix of the secondaspect for affinity purification of immunoglobulins or variants thereof,i.e. the Fc binding protein of the invention is used for affinitychromatography. In some embodiments, the Fc binding protein of theinvention is immobilized onto a solid support as described in the secondaspect of the invention.

Method of Affinity Purification of Immunoglobulins.

In a fourth aspect the present invention is directed to a method ofaffinity purification of immunoglobulins, the method comprising (a)providing a liquid containing an immunoglobulin; (b) providing anaffinity separation matrix comprising an immobilized Fc binding proteinof the first aspect coupled to said affinity separation matrix; (c)contacting said liquid with said affinity separation matrix, whereinsaid immunoglobulin binds to said immobilized Fc binding protein; and(d) eluting said immunoglobulin from said matrix, thereby obtaining aneluate containing said immunoglobulin. In some embodiments, the methodof affinity purification may further comprising one or more washingsteps carried out between steps (c) and (d) under conditions sufficientto remove from the affinity separation matrix some or all molecules thatare non-specifically bound thereto. Non-specifically bound means anybinding that does not involve an interaction between the at least onebinding domain of the presently disclosed subject matter and anImmunoglobulin. Affinity separation matrixes suitable for the discloseduses and methods are those matrixes according to the embodimentsdescribed above and as known to someone skilled in the art.

In some embodiments of the fourth aspect, the elution of theimmunoglobulin from the matrix in step (d) is effected through a changein pH and/or a change in salt concentration. Any suitable solution canbe used, for example by a solution with pH 5 or lower (see e.g. Table 3in Example 9), or by a solution with pH 11 or higher.

In some embodiments, a further step (f) for efficient cleaning andsanitization of the affinity matrix is added, preferably by using analkaline liquid, for example, with pH of 13-14. In certain embodiments,the cleaning liquid comprises 0.1-1.0 M NaOH or KOH, preferably 0.25-0.5M NaOH or KOH. Due to the high alkaline stability of the Fc bindingproteins of the invention, such strong alkaline solution can be used forcleaning purposes.

In some embodiments, the affinity matrix can be re-used at least 10times, at least 20 times, at least 30 times, at least 40 times, at least50 times, at least 60 times, at least 70 times, at least 80 times, atleast 90 times, or at least 100 times, due to a repetition of steps (a)to (e), optionally (a) to (f) can be repeated at least 10 times, atleast 20 times, at least 30 times, at least 40 times, at least 50 times,at least 60 times, at least 70 times, at least 80 times, at least 90times, or at least 100 times.

In general, suitable conditions for performing the method of affinitypurification are well known to someone skilled in the art. In someembodiments, the disclosed uses or methods of affinity purificationcomprising the disclosed Fc binding domains may provide elution of atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99% of Fc containing proteins ata pH of greater than or equal to 3.5 (e.g., about 4.0, about 4.5, about5.0, about 5.5, about 6.0, or about 6.5).

Nucleic Acid Molecule.

In a fifth aspect, the present invention is directed to a nucleic acidmolecule, preferably an isolated nucleic acid molecule, encoding a Fcbinding protein of any embodiment disclosed above. In one embodiment,the present invention is directed to a vector comprising the nucleicacid molecule. A vector means any molecule or entity (e.g., nucleicacid, plasmid, bacteriophage or virus) that can be used to transferprotein coding information into a host cell. In one embodiment, thevector is an expression vector.

In a sixth aspect, the present invention is directed to an expressionsystem which comprises a nucleic acid or a vector as disclosed above,for example a prokaryotic host cell, for example E. coli, or aeukaryotic host, for example yeast Saccharomyces cerevisiae or Pichiapastoris or mammalian cells such as CHO cells.

Method for the Production of a Fc Binding Protein.

In a seventh aspect the present invention is directed to a method forthe production of a Fc binding protein of the invention, comprising thestep(s): (a) culturing the host cell of the sixth aspect under suitableconditions for the expression of the binding protein in order to obtainsaid Fc binding protein; and (b) optionally isolating said Fc bindingprotein. Suitable conditions for culturing a prokaryotic or eukaryotichost are well-known to the person skilled in the art.

Fc binding molecules of the invention may be prepared by any of the manyconventional and well-known techniques such as plain organic syntheticstrategies, solid phase-assisted synthesis techniques or by commerciallyavailable automated synthesizers. On the other hand, they may also beprepared by conventional recombinant techniques alone or in combinationwith conventional synthetic techniques.

One embodiment of the present invention is directed to a method for thepreparation of a Fc binding protein according to the invention asdetailed above, said method comprising the following steps: (a)preparing a nucleic acid encoding an Fc binding protein as definedabove; (b) introducing said nucleic acid into an expression vector; (c)introducing said expression vector into a host cell; (d) cultivating thehost cell; (e) subjecting the host cell to culturing conditions underwhich an Fc binding protein is expressed, thereby (e) producing an Fcbinding protein as described above; optionally (f) isolating the proteinproduced in step (e); and (g) optionally conjugating the protein tosolid matrices as described above.

In a further embodiment of the present invention the production of theFc binding protein is performed by cell-free in vitrotranscription/translation.

EXAMPLES

The following Examples are provided for further illustration of theinvention. The invention, however, is not limited thereto, and thefollowing Examples merely show the practicability of the invention onthe basis of the above description.

Example 1. Generation of Artificial Mosaic Proteins that Bind to Fc

Fc binding proteins were initially generated by a shuffling process ofnaturally occurring Protein A domains (E, B, D, A, C, Z). In moredetail, the shuffling process as understood herein is an assemblyprocess resulting in artificial amino acid sequences starting from a setof non-identical known amino acid sequences. The shuffling processcomprised the following steps: a) providing sequences of five naturallyoccurring Protein A domains E, B, D, A, and C, and Protein A variantdomain Z; b) alignment of said sequences; c) statistical fragmentationin silico to identify subsequences that were recombined, and then d)assembly of new, artificial sequences of the various fragments toproduce a mosaic product, i.e. a novel and artificial amino acidsequence. The fragments generated in step c) were of any length, e.g. ifthe fragmented parent sequence had a length of n, the fragments was oflength 1 to n−1.

The relative positions of the amino acids in the mosaic products weremaintained with respect to the starting amino acid sequences. At least90% of positions Q9, Q10, A12, F13, Y14, L17, P20, L22, Q26, R27, F30,I31, Q32, S33, L34, D36, D37, P38, S39, S41, L45, E47, A48, K50, L51,Q55, A56, P57 are identical between the artificial mosaic amino acidsequences of for example IB14, IB27, IB24, IB26, IB28, IB20 (SEQ ID NOs:78-83) and naturally occurring Protein A domains or Protein A domainvariants. The mosaic domains and naturally occurring Protein A domainsor Protein A domain variants are characterized by three-helix bundles of58 amino acids with helix 1 from amino acid residues 7-19, helix 2 fromamino acid residues 23-37, and helix 3 from amino acid residues 40-55.The overall amino acid sequence of, for example, artificial Fc bindingdomains IB14, IB26, and IB27 is artificial in that it is not more than85% identical to the overall amino acid sequence of any of the naturallyoccurring Protein A domains or domain Z. After the initial artificial Fcbinding proteins was generated, in some cases the protein was furthermodified by site-specific randomization of the amino acid sequence tofurther modify biochemical properties. The further modifications wereintroduced by site-saturation mutagenesis of individual amino acidresidues.

Genes for artificial Fc binding proteins were synthesized and clonedinto an E. coli expression vector using standard methods known to askilled person. DNA sequencing was used to verify the correct sequenceof inserted fragments.

To generate multimeric Fc binding proteins, 2 or 3 identical Fc bindingdomains were genetically fused.

For specific purification, optionally a strep-tag (WSHPQFEK; SEQ ID NO:89) was added to the C-terminus of the Fc binding proteins.

Example 2. Mutagenesis to Generate Variants

For site-directed mutagenesis, the Q5® site-directed Mutagenesis Kit(NEB; Cat. No. E0554S) was used according to the manufacturer'sinstructions. PCRs were carried out with oligonucleotides coding foreach specific substitution respectively and a plasmid containing thetemplate. Products were ligated and transformed into E. coli XL2-bluecells (Stratagene) via electroporation. Single colonies were isolatedand DNA sequencing was used for insert containing clones to verify thecorrect sequences.

A combination of several point mutations was generated byGeneArt™Strings™ synthesis (Thermo Fisher Scientific). The Strings DNAfragments corresponded to a purified PCR product and were cloned into aderivate of a pET28a vector. Ligation products were transformed into E.coli XL2-blue cells via electroporation. Single colonies were screenedby PCR to identify constructs containing inserts of the right size. DNAsequencing was used to verify the correct sequences. Some variants withpoint mutations are shown for example in FIG. 1 .

Example 3. Expression of Fc Binding Proteins

BL21 (DE3) competent cells were transformed with an expression plasmidencoding Fc binding proteins. Cells were spread onto selective agarplates (Kanamycin) and incubated overnight at 37° C. Precultures wereinoculated from single colony in 100 ml 2×YT medium and cultured for 16hours at 37° C. at 160 rpm in a conventional orbital shaker in baffled 1L Erlenmeyer flasks supplemented with 150 μg/ml Kanamycin withoutlactose and antifoam. The OD₆₀₀ readout should be in the range of 6-12.Main culture was inoculated from previous overnight culture with anadjusted start-OD₆₀₀ of 0.5 in 400 ml superrich medium (modified H15medium 2% Glucose, 5% Yeast extract, 0.89% Glycerol, 0.76% Lactose, 250mM MOPS, 202 mM TRIS, pH 7.4, Antifoam SE15) in 1 L thick-walledErlenmeyer flasks that was supplemented with 150 μg/ml Kanamycin.Cultures were transferred to a resonant acoustic mixer (RAMbio) andincubated at 37° C. with 20×g. Aeration was facilitated by Oxy-Pumpstoppers. Recombinant protein expression was induced by metabolizingglucose and subsequently allowing lactose to enter the cells. Atpredefined time points OD₆₀₀ was measured, samples adjusted to 5/OD₆₀₀were withdrawn, pelleted and frozen at −20° C. Cells were grownovernight for approx. 24 hours to reach a final OD₆₀₀ of about 45-60. Tocollect biomass cells were centrifuged at 16000×g for 10 min at 20° C.Pellets were weighed (wet weight) and pH was measured in thesupernatant. Cells were stored at −20° C. before processing.

Example 4: SDS-PAGE Analysis of Expression and Solubility of Fc BindingProteins

Samples taken during fermentation were resuspended in 300 μl extractionbuffer (PBS supplemented with 0.2 mg/ml Lysozyme, 0.5× BugBuster, 7.5 mMMgSO₄, 40 U Benzonase) and solubilized by agitation in a thermomixer at700 rpm, rt for 15 min. Soluble proteins were separated from insolubleproteins by centrifugation (16000×g, 2 min, rt). Supernatant waswithdrawn (soluble fraction) and the pellet (insoluble fraction) wasresuspended in equivalent amount of urea buffer (8 M urea, 0.2 M Tris, 2mM EDTA, pH 8.5). 50 μl were taken both from the soluble and insolublefraction, and 12 μl 5× sample buffer as well as 5 μl 0.5 M DTT wereadded. Samples were boiled at 95° C. for 5 min. Finally, 8 μl of thosesamples were applied to NuPage Novex 4-12% Bis-Tris SDS gels which wererun in accordance to the manufacturer's recommendations and stained withCoomassie. High level expression of all Fc binding proteins was foundunder optimized conditions within the chosen period of time (data notshown). All expressed Fc binding proteins were soluble to more than 95%according to SDS-PAGE.

Example 5: Purification of Fc Binding Proteins

Fc binding proteins were expressed in the soluble fraction of E. coliwith a C-terminal StrepTagII (WSHPQFEK). The cells were lysed by twofreeze/thaw cycles and the purification step was performed withStrep-Tactin®-resin according to the manufacturer's instructions (IBA,Goettingen, Germany). To avoid disulfide formation the buffers weresupplemented with 1 mM DTT.

Alternatively, Fc binding proteins were expressed in the solublefraction of E. coli with a C-terminal StrepTagII. The cells wereresuspended in cell disruption buffer and lysed by a constant celldisruption system (Unit F8B, Holly Farm Business Park) at 1 kbar for twocycles. Purification step was performed with Strep-Tactin-resin (IBA,Goettingen, Germany) and additional gel filtration (Superdex 75 16/60;GE Healthcare) using an AKTAxpress system (Ge Healthcare) according tothe manufacturer's instructions. To avoid disulfide formation buffersfor Strep-Tactin-purification were supplemented with 1 mM DTT andcitrate-buffer (20 mM Citrat, 150 mM NaCl, pH 6.0) was used as runningbuffer for gel filtration.

Example 6. The Fc Binding Proteins Bind to IgG with High Affinities (asDetermined by ELISA)

The affinities of the Fc binding proteins towards IgG₁ or IgG₂ or IgG₄were determined using an Enzyme Linked Immunosorbent Assay (ELISA). IgG₁or IgG₂ or IgG₄ containing antibodies (e.g. Cetuximab for IgG₁,Panitumumab for IgG₂, or Natalizumab for IgG₄) were immobilized on a 96well Nunc MaxiSorb ELISA plate (2 μg/ml). After incubation for 16 h at4° C. the wells were washed three times with PBST (PBS+0.1% Tween 20)and the wells were blocked with 3% BSA in PBS (2 h at room temperature).The negative controls were wells blocked only with BSA. After blocking,the wells were washed three times with PBST and incubated for 1 h withthe Fc binding protein (in PBST) at room temperature. After incubationthe wells were washed three times with PBST and subsequently incubatedwith Strep-Tactin-HRP (1:10000) (IBA, Goettingen, Germany) for 1 h atroom temperature. Afterwards the wells were washed three times with PBSTand three times with PBS. The activity of the horseradish peroxidase wasvisualized by adding TMB-Plus substrate. After 30 min the reaction wasstopped by adding 0.2 M H₂SO₄ and the absorbance was measured at 450 nm.For example, as determined via ELISA, the K_(D) for human IgG₁ is 4.9 nMfor IB14; 3.4 nM for domain Z; 3.1 nM for domain B; and 2.8 nM fordomain C.

Example 7. The Fc Binding Proteins Bind to IgG with High Affinities (asDetermined with Surface Plasmon Resonance Experiments)

A CM5 sensor chip (GE Healthcare) was equilibrated with SPR runningbuffer. Surface-exposed carboxylic groups were activated by passing amixture of EDC and NHS to yield reactive ester groups. 700-1500 RUon-ligand were immobilized on a flow cell, off-ligand was immobilized onanother flow cell. Injection of ethanolamine after ligand immobilizationremoves non-covalently bound Fc binding protein. Upon ligand binding,protein analyte was accumulated on the surface increasing the refractiveindex. This change in the refractive index was measured in real time andplotted as response or resonance units (RU) versus time. The analyteswere applied to the chip in serial dilutions with a suitable flow rate(μl/min). After each run, the chip surface was regenerated withregeneration buffer and equilibrated with running buffer. The controlsamples were applied to the matrix. Regeneration and re-equilibrationwere performed as previously mentioned. Binding studies were carried outby the use of the Biacore® 3000 (GE Healthcare) at 25° C.; dataevaluation was operated via the BIAevaluation 3.0 software, provided bythe manufacturer, by the use of the Langmuir 1:1 model (RI=0). Evaluateddissociation constants (K_(D)) were standardized against off-target andK_(D) values of different artificial Fc binding proteins for humanIgG₁-Fc, Cetuximab (IgG₁), Natalizumab (IgG₄), or Panitumomab (IgG₂) areshown in Table 1.

TABLE 1 K_(D) values of Fc binding proteins for Ig Fc binding SEQ ID Kd[nM] Kd [nM] Kd [nM] Kd [nM] protein NO: IgG-Fc IgG₁ IgG₄ IgG₂ IB14 781.28 IB14 E43C 74 45.5 IB14 G46C 75 72.9 IB14 E47C 76 48.8 IB14 K50C 7742.8 cs14 3 6.48 2.9 2.51 7.42 cs14 G46C 18 4.88 3.38 35.6 cs14 E43C 170.453 0.302 1.75 cs27 4 3.64 2.54 21.6 cs27 G46C 22 0.518 0.302 2.74cs26 7 6.5 5.81 66.6 cs26 A46C 27 5.75 3.00 101 cs26 A46C 32 2.59 1.8620.5 (dimer) cs26 A46C 35 2.23 1.98 13.3 (trimer) Domain C 84 4.91 3.9558.1

Example 8. Fc Binding Proteins Coupled to an Epoxy-Activated Matrix(Sepharose 6B)

Purified Fc binding proteins were coupled to epoxy-activated matrix(Sepharose 6B, GE; Cat. No. 17-0480-01) according to the manufacturer'sinstructions (coupling conditions: pH 9.0 overnight, blocking for 5 hwith ethanolamine). Cetuximab was used as IgG sample (5 mg; 1 mg/mlmatrix). Cetuximab was applied in saturated amounts to the matrixcomprising immobilized Fc binding protein. The matrix was washed with100 mM glycine buffer, pH 2.5 to elute Cetuximab that was bound to theimmobilized IgG-binding protein. The concentration of the eluted IgG wasmeasured by BLI (quantification with Protein A Octet-sensors andCetuximab as standard) in order to determine the binding activity of theFc binding proteins. FIG. 2A shows the coupling efficiency of 1614 (SEQID NO: 78) and 1614 variants with Cystein in Positions 43, 46, 47, 50,or 58 (SEQ ID NOs: 74-77, respectively). The coupling efficiency of allIB14 variants with Cys in positions 43, 46, 47, 50, or 58 was higherthan for IB14. FIG. 2B shows the coupling of cs14 46C (SEQ ID NO: 18) orcs14 43C (SEQ ID NO: 17) to Epoxy activated Sepharose 6B matrix comparedto cs14 with an C-terminal Cys and compared to a commercially availableProtein A. Coupling conditions were 450 μM, 4500 nmol/ml, pH 9, 2 h or18 h at 30° C., 1 mM TCEP, 1 M (NH₄)₂SO₄. Only minor change in netcoupling rate were observed between 2 h and 18 h. Coupling rates areshown in nmole (domain)/ml.

Example 9. Alkaline Stability of Fc Binding Proteins Coupled to anEpoxy-Activated Matrix

Columns were incubated with 0.5 M NaOH for 0 h, 6 h, 18 h, 24 h, 36 h,or 72 h at room temperature (22° C.+/−3° C.). The Ig binding activity ofthe immobilized proteins was analyzed before and after incubation with0.5 M NaOH. The Ig binding activity of immobilized proteins before NaOHtreatment was defined as 100%. The remaining IgG binding activity aftercontinuous 0.5 M NaOH treatment for 6 hours is shown in FIG. 3A for 1614variants (SEQ ID NOs: 74-77). FIG. 3B shows caustic stability aftercontinuous 0.5 M NaOH treatment for 0 h, 18 h, or 72 h for cs14 46C,cs14 43C, cs14, and for a commercially available Protein A. The proteinswere immobilized to Epoxy Sepharose for 2 h at 30° C. (4500 nmol/ml).The dynamic binding capacity DBC 10% was determined at 5 min residencetime. Cs14 46C shows the highest DBC 10% after 72 h of continuous 0.5NaOH treatment for 19.5 mg/ml which is more than 20% more than the valuemeasured for the commercially available Protein A. FIG. 4 shows theremaining binding capacity after continuous 0.5 M NaOH treatment for 6,24, and 36 hours for cs26 46C (SEQ ID NO: 27) compared to cs26 (SEQ IDNO: 7) and to wildtype domain C (SEQ ID NO: 84). The binding capacity ofcs26 A46C (monomer or dimer) remains >20% after at least after 36 h 0.5M NaOH incubation, see Table 2.

TABLE 2 Caustic stability of cs26 A46C compared to cs26 and to wildtypedomain C Fc binding protein 0 h 6 h 24 h 36 h vs C @ 36 h cs26 A46C 10095 83 78  42% cs26 A46C (dimer) 100 92 81 72 24.8% cs26 100 105 82 7327.7% domain C 100 95 76 62

Example 10. Fc Binding Proteins Coupled to Agarose-Based ChromatographyBeads Praesto™ Pure45

Purified Fc binding proteins were coupled to agarose-basedchromatography beads (Praesto™ Pure45, Purolite; Cat. No. PR01262-166)according to the manufacturer's instructions (coupling conditions: pH9.5, 3 hours, 35° C., 4.1 M NaSO₄, blocking overnight withethanolamine). Polyclonal human IgG Gammanorm® (Ocatpharm) was used asIgG sample (conc. 2.2 mg/ml). Polyclonal hIgG sample was applied insaturated amounts to the matrix comprising immobilized Fc bindingprotein. The matrix was washed with 100 mM Citrate buffer, pH 2.0 toelute hIgG that was bound to the immobilized Fc binding protein. Dynamicbinding capacity was determined for cs26 46C (monomer and dimer; (SEQ IDNOs: 27, 32) compared to recombinant wildtype Protein A by the mass ofinjected hIgG at 10% breakthrough at 6 min residence time. FIG. 5 showsthat Cs26 46C has a 61.2% higher DBC at 6 min residence time thanrecombinant Protein A.

Example 11. Elution of IgG from Fc Binding Proteins Coupled toAgarose-Based Chromatography Beads Praesto™ Pure45 and/or Pure85

Purified Fc binding proteins (cs26 46C) were coupled to agarose-basedchromatography beads (Praesto™ Pure45 or Pure 5) according to themanufacturer's instructions. Polyclonal human IgG Gammanorm® andmonoclonal IgG₁ antibody Cetuximab was used as IgG sample (conc. 2.2mg/ml), loading up to DBC10%. Polyclonal hIgG sample was applied insaturated amounts to the matrix comprising immobilized Fc bindingprotein. In a two-step process, the matrix was first washed with 100 mMCitrate buffer, pH 3.5 and then with 100 mM Citrate buffer, pH 2.0 toelute hIgG that was bound to the immobilized Fc binding protein. Table 3shows that almost 100% of the bound IgG was eluted from beads coupledwith cs26 46C at pH 3.5.

TABLE 3 Elution of IgG from cs26 46C coupled to beads Eluted protein atEluted protein at Recovery: 0.1M citrate pH 3.5 0.1M citrate pH 2.0Load/Elution Antibody (%) (%) (%) Gammanorm 99.8 0.2 85 Cetuximab 100 086

The invention claimed is:
 1. An Fc binding protein comprising one ormore domains, wherein the one or more domains comprises an amino acidsequence of any one of SEQ ID NOS: 3-86, 90-99 or an amino acid sequencewith at least 89.5% identity to one of said sequences and wherein atleast one amino acid in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or54 corresponding to SEQ ID NO: 3 is cysteine.
 2. The Fc binding proteinof claim 1, wherein at least one domain comprises an amino acid sequenceof SEQ ID NO: 3 or an amino acid sequence with at least 89.5% identitythereto wherein at least one amino acid in position 40, 42, 43, 46, 47,49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3 is cysteine.
 3. TheFc binding protein of claim 1, wherein at least one domain comprises anamino acid sequence of SEQ ID NOs: 3-16 or an amino acid sequence withat least 89.5% identity thereto, respectively, wherein at least oneamino acid in position 40, 42, 43, 46, 47, 50, 51, 53, or 54corresponding to SEQ ID NOs: 3-16 is cysteine.
 4. The Fc binding proteinof claim 1, wherein at least one domain comprises an amino acid sequenceof SEQ ID NOs: 7-8 or an amino acid sequence with at least 89.5%identity thereto, respectively, wherein at least one amino acid inposition 40, 42, 43, 46, 47, 50, 51, 53, or 54 corresponding to SEQ IDNOs: 7-8 is cysteine.
 5. The Fc binding protein of claim 1, wherein atleast one amino acid in position 43, 46, or 47 corresponding to SEQ IDNO: 3 is cysteine.
 6. The Fc binding protein of claim 1, wherein atleast one domain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 17-86, 90-99, or an amino acid sequencewith at least 89.5% identity thereto, respectively.
 7. The Fc bindingprotein of claim 1, wherein said domain has a deletion of 1, 2, or 3amino acids within the first 4 amino acids of its N-terminus and/or adeletion of 1 or 2 amino acids within the first 2 amino acids of itsC-terminus.
 8. The Fc binding protein of claim 1, wherein the proteincomprises 2, 3, 4, 5, 6, 7, or 8 domains linked to each other.
 9. The Fcbinding protein of claim 8, wherein the protein is a homo-multimer or ahetero-multimer.
 10. The Fc binding protein of claim 9, wherein one ormore domains are linked to each other directly or with one or morelinkers.
 11. The Fc binding protein of claim 1, wherein the protein isconjugated to a solid support.
 12. The Fc binding protein of claim 1,wherein said protein binds to IgG1, IgG2, IgG4, IgM, IgA, Ig fragmentscomprising an Fc region, fusion proteins comprising an Fc region of anIg, and conjugates comprising an Fc region of an Ig.
 13. An affinityseparation matrix comprising an Fc binding protein comprising one ormore domains, wherein the one or more domains comprises an amino acidsequence of any one of SEQ ID NOS: 3-16 or an amino acid sequence withat least 89.5% identity to one of said sequences and wherein at leastone amino acid in position 40, 42, 43, 46, 47, 49, 50, 51, 53, or 54corresponding to SEQ ID NO: 3 is cysteine.
 14. The affinity separationmatrix of claim 13, wherein at least one domain comprises an amino acidsequence of SEQ ID NO: 3 or an amino acid sequence with at least 89.5%identity thereto wherein at least one amino acid in position 40, 42, 43,46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3 is cysteine.15. The affinity separation matrix of claim 13, wherein at least onedomain comprises an amino acid sequence of SEQ ID NOs: 7-8 or an aminoacid sequence with at least 89.5% identity thereto, respectively,wherein at least one amino acid in position 40, 42, 43, 46, 47, 50, 51,53, or 54 corresponding to SEQ ID NOs: 7-8 is cysteine.
 16. The affinityseparation matrix of claim 13, wherein at least one amino acid inposition 43, 46, or 47 corresponding to SEQ ID NO: 3 is cysteine.
 17. Amethod of affinity purification of a protein comprising an Fc sequence,the method comprising: (a) providing a liquid that contains proteincomprising an Fc sequence; (b) providing an affinity separation matrixcomprising an Fc binding protein comprising one or more domains, whereinthe one or more domains comprises an amino acid sequence of any one ofSEQ ID NOS: 3-16 or an amino acid sequence with at least 89.5% identityto one of said sequences and wherein at least one amino acid in position40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3is cysteine, wherein the Fc binding protein is coupled to said affinityseparation matrix; (c) contacting said affinity separation matrix withthe liquid under conditions that permit binding of the Fc bindingprotein to a protein comprising an Fc sequence; and (d) eluting saidprotein comprising an Fc sequence from said affinity separation matrix.18. The method of claim 17, wherein at least one domain comprises anamino acid sequence of SEQ ID NO: 3 or an amino acid sequence with atleast 89.5% identity thereto wherein at least one amino acid in position40, 42, 43, 46, 47, 49, 50, 51, 53, or 54 corresponding to SEQ ID NO: 3is cysteine.
 19. The method of claim 17, wherein at least one domaincomprises an amino acid sequence of SEQ ID NOs: 7-8 or an amino acidsequence with at least 89.5% identity thereto, respectively, wherein atleast one amino acid in position 40, 42, 43, 46, 47, 50, 51, 53, or 54corresponding to SEQ ID NOs: 7-8 is cysteine.
 20. The method of claim17, wherein at least one amino acid in position 43, 46, or 47corresponding to SEQ ID NO: 3 is cysteine.
 21. The Fc binding protein ofclaim 10, wherein said one or more linkers is a peptide linker.
 22. TheFc binding protein of claim 11, wherein the protein is conjugated to asolid support via a cysteine in position 40, 42, 43, 46, 47, 50, 51, 53,or 54.